Chilling-resistant plants and their production

ABSTRACT

The present invention relates to the genetic engineering of higher plants to confer chilling resistance. Provided is a higher plant which contains more unsaturated fatty acids in membrane lipids than are inherent to that plant species, and a process for producing the same. A preferred embodiment of such plant is a transgenic plant expressing a polypeptide with a glycerol 3-phosphate acyltransferase activity that has a higher substrate selectivity for oleoyl-(acyl-carrier-protein) (oleoyl-ACP) than for palmitoyl-(acyl-carrier-protein) (palmitoyl-ACP). In another aspect, there are provided higher plants with a lowered critical temperature for chilling injury, and a process to produce the same. A preferred embodiment of such plant is a transgenic plant whose phosphatidylglycerol contains reduced amount of saturated molecular species due to the expression of a polypeptide with a glycerol 3-phosphate acyltransferase activity that has a higher substrate selectivity for oleoyl-ACP than for palmitoyl-ACP.

FIELD OF THE INVENTION

The present invention relates to a plant with an altered fatty acidcomposition of lipids, more specifically, a plant made resistant tochilling injury by altering the fatty acid composition of its lipids,and a process to produce such plant.

BACKGROUND OF THE INVENTION

Low temperature injury of higher plants is largely categorized into twodifferent types. One is the injury caused by temperatures at or below 0°C. and is called "freezing injury". The other, which is the subjectmatter of the present invention, is called "chilling injury" and istotally different from freezing injury. Most tropical and subtropicalplants suffer chilling injury at temperatures in the range of 5° to 15°C., which injury damages the tissue(s) of whole and/or a part of theplants leading to a variety of physiological dysfunctions and ultimatelyto death in the severest cases.

Plants susceptible to chilling injury are called "chilling-sensitive"plants and include many important crops such as rice, maize, yam, sweetpotato, cucumber, green pepper, eggplant, squash, banana, melon,kalanchoe, cyclamen, lily, rose, castor bean, sponge cucumber andtobacco. These plants suffer a variety of injuries, such as theinhibition of germination and growth, tissue necrosis as well as thedeath of the whole plant, at temperatures between 5° and 15° C., in mostcases at around 10° C., and thus are prone to damage by cold weather andfrost. Furthermore, fruits, vegetables, and the like harvested fromchilling-sensitive plants cannot tolerate low temperature storage (asillustrated by the black decaying spots that quickly appear on bananaswhen taken out of a refrigerator) making it difficult to store theseharvests for a long period after the harvest.

Most plants of temperate origin, on the other hand, arechilling-resistant and are not injured even by a low temperature ofaround 0° C. Chilling-resistant crop plants include wheat, barley,spinach, lettuce, radish, pea, leek, and cabbage. Wild weeds such asdandelion and Arabidopsis are also chilling-resistant.

Chilling injury is significantly related to the fluidity of membranelipids that constitute biomembranes. Biomembranes are one of basicorganizing units of living cells. They define the inside and outside ofcells as the cell membrane and in eukaryotic cells also organize avariety of membrane structures (cell organelles) to partition the cellinto several functional units. Biomembranes are not mere physicalbarriers against high molecular weight substances and low molecularweight electrolytes; the function of proteins associated with themembranes allow the selective permeation, and/or the active transportagainst concentration gradient, of particular substances. In this waybiomembranes keep the micro-environment of cytoplasm and cell organellesin a suitable condition for their purpose. Some biochemical processes,such as energy production by respiration and photosynthesis, require aspecific concentration gradient of particular substances acrossbiomembranes. In photosynthesis, the energy of light generates ahydrogen ion gradient potential across the thylakoid membrane withinchloroplasts, which potential energy is then convened to ATP, ahigh-energy compound utilized by living cells, by proteins in thethylakoid membrane. Accordingly, if biomembranes fail to function as abarrier as described above, it will disturb not only themicro-environment of cells but impair these cellular functions based ona concentration gradient, leading to serious dysfunctions of livingcells.

The membrane lipids that constitute biomembranes are mainlyphospholipids and, in the case of chloroplasts, glycerolipids.Phospholipids are 1,2-di long-chain alkyl (fatty acyl) esters ofglycerol with a polar group bonded at the 3 position as a phosphoester.They are amphipathic compounds having both a hydrophilic portion (thepolar group) and a hydrophobic portion (the fatty acyl groups) withinone molecule and therefore form a lipid bilayer with the hydrophobicportions inside and the hydrophilic portions on the surface whendispersed in an aqueous solution. This lipid bilayer is the basicstructure of biomembranes which "buries" a variety of proteins insideand/or on its surface. Under physiological conditions, the lipid bilayeris in the liquid-crystalline phase in which the inside of the bilayerretains a high fluidity, allowing free horizontal dispersion androtation of protein and lipid molecules within the membrane. Thisfluidity of biomembranes is essential for cellular functions (Darnell,J. et al, Molecular cell biology, Scientific American Books, 1986).

When the temperature of a simple lipid bilayer in the liquid-crystallinephase is lowered to a certain temperature called the phase transitiontemperature (Tc), the bilayer undergoes a phase transition to the gelphase in which the inside of the membrane has less fluidity. In the caseof biomembranes, which consist of different types of lipids, some lipidswith a high Tc begin to form gel phase domains at a certain temperaturewhile other lipids with a lower Tc are still in the liquid-crystallinephase, resulting in the phase separation, in which both theliquid-crystalline and gel phases co-exist. In a phase separated state,biomembranes become leaky and no more serve as a barrier against lowmolecular weight electrolytes.

A relationship between chilling injury and the phase transition ofmembrane lipids was first proposed in early 1970's (Lyons, J. M., Ann.Rev. Plant Physiol., 24:445, 1973). At that time, however, there was noconcrete data supporting the existence of the relationship. Later, in aseries of experiments using cyanobacteria (blue-green algae) as modelorganisms, it was shown that the chilling injury of cyanobacteria is theresult of irreversible effluent from the cells of electrolytes such asions following the phase separation of the cell membrane at a chillingtemperature (Murata, N. and Nishida, I., in The biochemistry of plantsvol. 9 Lipids: Structure and function, p.315, Academic Press, Orlando,1987).

Lipids are generally classified by the polar group (see above for thestructure of membrane lipids), since their behavior in column and thinlayer chromatographics is largely determined by the polar group. Amongone particular class of lipids with the same polar group, there are manydifferent molecules with various combinations of the two fatty acylgroups in the molecule. The term "molecular species" is used todistinguish these molecules. The Tc of each lipid molecular speciesdepends on the polar group as well as the chain length and degree ofunsaturation (the number of double bonds) of the fatty acyl groups, andin some instances the environmental salt concentration and such. Amongthese, the degree of unsaturation of the fatty acyl groups has thelargest effect; while a particular molecular species with two saturatedfatty acyl groups usually has a Tc above room temperature, introductionof only one double bond into one of the fatty acyl groups results in thedecrease of Tc to around 0° C. (Santaten, J. F. et al, Biochem. Biophys.Acta, 687:231, 1982). (However, if the double bond is in the transconfiguration, the effect on the Tc is very small [Phillips, M. C. etal, Chem. Phys. Lipids, 8:127, 1972]. Most double bonds of membranelipids are in the cis configuration and the trans configuration isrelatively rare.) This indicates that a lipid molecular species with atleast one double bond in its fatty acyl groups (hereinafter called"unsaturated molecular species") does not undergo phase transition ataround 10° C., the critical temperature for chilling injury.Consequently, only those lipid molecules with two saturated fatty acylgroups (hereinafter called "saturated molecular species") could inducethe phase separation of biomembranes which is considered to be theprimary event in chilling injury.

Membrane lipids have been extracted from several chilling-sensitive andresistant plants, separated according to the polar group, and theirfatty acid and molecular species compositions analyzed. The resultsshowed that only phosphatidylglycerol (PG) contains a significant amountof saturated molecular species among plant membrane lipids and that thecontent of saturated molecules in PG is high (30-70 %) inchilling-sensitive plants and low (<20 %) in chilling-resistant plants(Murata, N., Plant Cell Physiol, 24:81, 1983; Roughan, P. G., PlantPhysiol., 77:740, 1985). Since PG is a major component of plastid(chloroplast, chromoplast) biomembranes, this correlation between the PGmolecular species composition and chilling sensitivity strongly suggeststhat the primary event in the chilling injury of higher plants is thephase separation of plastid biomembranes induced by the phase transitionof PG (Murata, N. and Nishida, I., in Chilling injury of horticulturalcrops, p.181, CRC Press, Boca Raton, 1990).

PG is localized in plastids and, in the case of green leaves,synthesized mainly in chloroplasts (Sparace, S. A. and Mudd, J. B.,Plant Physiol., 70:1260, 1982). Its biosynthesis follows the steps shownbelow.

1. Transfer of a fatty acyl group to the sn-1 position of glycerol3-phosphate.

2. Transfer of another fatty acyl group to the sn-2 position.

3. Esterification of glycerol to the 3-phosphate group.

4. Desaturation of fatty acyl groups on the molecule.

Fatty acids are exclusively synthesized in chloroplasts. The synthesizedfatty acids are supplied to steps 1 and 2 of PG synthesis as acyl-ACPcomplexes wherein the fatty acids are bound to a protein called acylcarrier protein (ACP). Most of the fatty acids synthesized inchloroplasts are palmitic acid (saturated C-16 acid, hereinafterdesignated as 16:0) and oleic acid (mono-unsaturated C-18 acid,hereinafter designated as 18:1).

Step 1 of the above scheme is catalyzed by acyl-ACP:glycerol 3-phosphateacyltransferase (EC 2.3.1.15) (hereinafter called ATase). This enzyme isa soluble enzyme in chloroplast stroma. It has been partially purifiedfrom spinach and pea (Bertrams, M. and Heinz, E., Plant Physiol.,68:653, 1981) and purified to homogeneity from squash (Nishida, I. etal, Plant Cell Physiol., 28:1071, 1987). It is encoded by a nucleargene, which has been cloned from squash, Arabidopsis and recently frompea (Ishizaki, O. et al, FEBS Lett., 238:424, 1988; Nishida, I. et al.,in Plant lipid biochemistry, structure and utilization, Portland Press,London, 1990; Weber, S. et al, Plant Molec. Biol., 17:1067, 1991).ATases from different sources differ in selectivity for the substrate,acyl-ACP. While ATascs from spinach, pea and Arabidopsis, which arcchilling-resistant, have a high selectivity for 18:1-ACP, ATase fromsquash, a chilling-sensitive plant, equally utilizes both 18:1-ACP and16:0-ACP (Frentzen, M. et al, Eur. J. Biochem., 129:629, 1983; Frentzen,M. et al., Plant Cell Physiol, 28:1195, 1988 ).

The enzyme that catalyzes step 2 of the above scheme is a membrane-boundenzyme of chloroplast envelope and utilizes only 16:0-ACP (Frentzen, M.et al, Eur. J. Biochem., 129:629, 1983). In a number of plant speciescalled 16:3 plants, the intermediate product of steps 1 and 2,phosphatidic acid (1,2-diacylglyccrol 3-phosphate), is also anintermediate compound for the biosynthesis of glycerolipids (mono- anddigalactosyldiacylglyccrols and sulfoquinovosyl-diacylglyccrol)synthesized in chloroplasts. Steps 1 and 2 are therefore common to thelipid biosynthesis in chloroplasts of the 16:3 plants.

Very little is known about the enzymes for steps 3 and 4 of PGbiosynthesis. However, it is well known that the desaturation of fattyacyl groups in PG is asymmetric. At the sn-1 position, most of 18:1 isfurther desaturated to have two or three double bonds while 16:0 is notdesaturated. At the sn-2 position, some of the bound 16:0 is desaturatedto 3-trans-hexadecenoic acid (hereinafter designated as 16:1t) but nocis-double bond is introduced. Since a trans-double bond is much lesseffective in decreasing the phase transition temperature, the conversionof 16:0 to 16:1t at tic position 2 of PG would decrease the Tc by onlyabout 10° C., so that the Tc is still higher than the criticaltemperature for chilling injury (Bishop, D. G. and Kenrick, J. R.,Phytochemistry, 26:3065, 1987). PG molecular species with 16:1t areaccordingly included within saturated molecular species hereinafter.Because no cis-double bond is introduced in the fatty acyl group atposition 2, the fatty acyl group at the position 1 is very important indetermining the content of saturated molecular species.

Chilling-sensitive crop plants suffer significant disadvantages inlow-temperature tolerance and long-term post-harvest storage asdescribed above. Nevertheless, many of chilling-sensitive crops are veryimportant and indispensable for agricultural production; for example,rice and maize are the main cereal crops in many parts of the world. Animprovement in the chilling resistance of these crops would make iteasier to grow them in a chilling environment and/or to store theirharvest for a long period. In the case of ornamental flowers andvegetables grown in a greenhouse due to their chilling-sensitivity,improvement of chilling resistance would make the greenhouse unnecessaryor save the heating expense to a great extent. Furthermore, theimprovement might expand the area where the crop is grown, sincetemperature is often the main factor to define the borders of cropdevelopment.

There is thus a significant demand for chilling resistant plants andchilling resistance has been one of the major goals of crop breeding.However, conventional crossing breeding is limited in genetic sourcesfor this purpose, because one can cross the crop only within the samespecies. Recent progress in genetic engineering of higher plants hasmade it possible to introduce genetic information into crops from anunlimited range of genetic sources. The application of geneticengineering to providing chilling-resistance would therefore beinvaluable.

As already described, the primary event in the chilling injury of higherplants is the phase separation of plastidial membranes, and theplastidial membranes of chilling-sensitive plants contain a higher mountof the saturated PG molecular species considered to induce the phaseseparation. It was thus suggested that it might be possible to increasethe chilling resistance of chilling-sensitive plants by changing thefatty acid composition of their PG to decrease the content of saturatedmolecular species (Murata, N., Plant Cell Physiol, 24:81, 1983).However, this was only a hypothesis and, to date, there has been noreport of any method to change the fatty acid composition of cellularlipids nor any report of a plant with an altered fatty acid composition.

SUMMARY OF THE INVENTION

The present invention provides novel methods for increasing theunsaturated fatty acid content of membrane lipids, particularlyphosphatidylglycerol (PG), in higher plant. Briefly summerized, thesemethods involve introducing and expressing a DNA sequence encoding apolypeptide with a glycerol 3-phosphate acyltransferase (ATase) activityhaving a higher substrate selectivity for oleoyl-(acyl-carrier-protein)(18:1-ACP) than for palmitoyl-(acyl-carrier-protein) (16:0-ACP).

The present invention also provides for higher plants which contain moreunsaturated fatty acids in membrane lipids than are inherent to plantsof the species. A preferred embodiment of such a plant is a transgenicplant expressing a polypeptide with a glycerol 3-phosphateacyltransferase activity that has a higher substrate selectivity foroleoyl-(acyl-carrier-protein) than for palmitoyl-(acyl-carrier-protein).

As previously noted, ATase catalyzes the first step of lipidbiosynthesis in chloroplasts and ATases from different plant speciesexhibit different substrate selectivity for acyl-ACPs. ATases fromchilling-resistant plants such as spinach, pea and Arabidopsis have ahigh selectivity for 18:1-ACP, and ATases from chilling-sensitive plantssuch as squash equally utilize both 18:1-ACP and 16:0-ACP.

DNA sequences for use in the present invention and encoding an ATasethat having a higher substrate selectivity for 18:1-ACP can be any ofthe DNA sequences encoding an ATase of a chilling-resistant plant andDNA sequences encoding ATases from organisms other than higher plants.Preferably, a DNA sequence encoding an ATase of a chilling-resistantplant, more preferably the ATase of spinach, pea or Arabidopsis, isemployed. The expression of the exgenous DNA sequence and the productionthereby of the ATase can be accomplished by providing the DNA sequencewith an appropriate combinations of expression regulatory sequences(promoter, terminator, and such) and a sequence encoding a transitpeptide necessary, for the transport of proteins into chloroplasts. TheDNA construct can be introduced into plant genome by any of theconventional techniques known to those skilled in art and suitable foruse with the target plant.

According to the present invention, introduction and expression in ahigher plant of an exogenous DNA sequence encoding an ATase that has ahigher substrate selectivity for 18:1-ACP increases the unsaturatedfatty acid content in membrane lipids of the plant. Of particularsignificance to practice of the invention is the increases in theunsaturated fatty acid content of PG resulting in a prominent decreaseof saturated PG molecular species. As already described, saturated PGmolecular species induce the phase separation of plastidialbiomembranes, and it has been shown that resistance to chilling injuryis inversely correlated to the content of saturated PG molecular specieswithin a particular plant species.

Consequently, in another aspect of the present invention, there isprovided a process to lower the critical temperature for chilling injuryof a higher plant species that is inherently injured by a lowtemperature above 0° C. (a chilling-sensitive plant) by decreasing thecontent of saturated phosphatidylglycerol molecular species.

Yet another aspect of the present invention provides higher plants withan improved resistance to chilling injury. In other words, the presentinvention provides inherently chilling-sensitive species of higherplants with a lowered critical temperature for chilling injury. Apreferred embodiment of such plant is a transgenic plant whosephosphatidylglycerol contains a reduced amount of saturated molecularspecies due to the expression of a polypeptide with a glycerol3-phosphate acyltransferase activity that has a higher substrateselectivity for oleoyl-(acyl-carrier-protein) than forpalmitoyl-(acyl-camer-protein).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a result of Western blotting analysis of Arabidopsis ATasetransgenic tobacco plants using an anti-Arabidopsis ATase antibody. Lane1 contains 50 ng of an Arabidopsis ATase preparation expressed in E.coli. Lane 2 contains 10 μg of the total chloroplast protein of anon-transformed control plant. Lanes 3-7 contain 10 μg each of the totalchloroplast protein from transgenic plants No. 1-5, respectively. Lane 8contains 10 μg of the total leaf protein from transgenic plant No. 1.

FIG. 2 shows the effect of a chilling treatment on the photosyntheticactivity of transgenic tobacco plants. From top to bottom: tobaccotransformed with the vector pBI121, the Arabidopsis ATase cDNA and thesquash ATase cDNA.

FIGS. 3A-3D show the effect of a chilling treatment on transgenictobacco plants at the whole plant level. Upper and lower plates arecontrol pBI-121 transformed tobacco plants and the Arabidopsis ATasecDNA transgenic plants, respectively, before (left) and after (right)the chilling treatment.

DETAILED DESCRIPTION OF THE INVENTION

In general, when a DNA sequence is to be expressed to produce thepolypeptide it encodes, expression regulatory sequences are essential inaddition to the coding sequence corresponding to the polypeptide.Particularly important are a promoter sequence upstream andpolyadenylation signals downstream of the coding sequence. In thepresent invention, any appropriate combination of promoters andpolyadenylation signals that are known to function in plant cells can beemployed; e.g. cauliflower mosaic virus 35S promoter, nopaline synthasepromoter, and ribulose bisphosphate carboxylase/oxygenase small subunitpromoter, as well as nopaline synthase polyadenylation signals andoctopine synthase polyadenylation signals. Furthermore, if the expressedpolypeptide is to be transported into a particular compartment of thecell, such as the chloroplasts, a transit or leader peptide sequence isnecessary at the N-terminus of the polypeptide. Accordingly, in thepresent invention, "DNA sequence encoding ATase (or a polypeptide withan ATase activity)" shall not be limited to the coding region butinclude the expression regulatory sequences and/or a DNA sequenceencoding the transit peptide.

A DNA sequence encoding a polypeptide with an ATase activity having ahigher substrate selectivity for 18:1-ACP than for 16:0-ACP suitable foruse in the present invention is preferably one encoding an ATase from achilling-resistant plant, particularly spinach, pea, or Arabidopsis.Such DNA sequences can be obtained in whole or part by chemicalsynthesis; alternatively and more preferably, the DNA sequence can beobtained by cloning a cDNA or genomic DNA encoding the ATase fromchilling-resistant plants. In the following examples, a cDNA sequenceencoding the ATase from Arabidopsis thaliana (Nishida, I. et al, inPlant lipid biochemistry, structure and utilization, Portland Press,London, 1990) was used. DNA sequences that can be used in thisinvention, however, are not limited to this particular cDNA sequence.

The nucleotide sequence of the cDNA encoding Arabidopsis ATase is shownin SEQ ID NO:1. The isolation of this cDNA itself is not a part of thepresent invention; nevertheless, a detailed process for its isolation isdescribed below in Experimental examples 1 and 2. Briefly, a genomicclone encoding Arabidopsis ATase was obtained from an Arabidopsisgenomic library using a cDNA fragment for squash ATase as the probe.This genomic DNA was then used to screen an Arabidopsis cDNA library toobtain the cDNA clone. The Arabidopsis cDNA of SEQ ID NO:1codes for apolypeptide of 459 amino acids (SEQ ID NO: 2) with a molecular mass of50,431. The N-terminal 90 amino acid portion of this polypeptide isassumed to be a transit peptide for the transport to chloroplasts whichis cleaved off during the transporting process, resulting in a matureenzyme of 369 amino acids. An ATase preparation from E. coil expressingthis cDNA has a higher substrate selectivity for 18:1 than for 16:0(Experimental example 3).

When the DNA sequence is a cDNA, appropriate expression regulatorysequences are necessary at upstream and downstream of the cDNA sequencein order to express the cDNA in transgenic plants. When the DNA sequenceis a genomic DNA fragment and contains regulatory sequences, thefragment may be used by itself. Furthermore, since the ATase expressedaccording to this invention is involved in the lipid biosynthesis ofchloroplasts, the ATase expressed in the transgenic plants must betransported from the cytoplasm to chloroplasts. Generally, a transitpeptide sequence at the N-terminus is necessary to transport anuclear-encoded protein to chloroplasts (Van den Broeck et al, Nature,313:358, 1985). Because plastidial ATases of higher plants are producedin the cytosol and function in chloroplasts by nature, DNA sequencesencoding the ATase from higher plants, be it a cDNA or a genomic DNAfragment, should contain a DNA sequence encoding an amino acid sequencefunctioning as a transit peptide, as illustrated by the cDNA forArabidopsis ATase used in the following examples. Nevertheless, ifnecessary, DNA sequences encoding a known transit peptide, such as thatof ribulose bisphosphate carboxylase/oxygenase small subunit, may beemployed.

The DNA sequence encoding a polypeptide (SEQ ID Nos: 1 and 2) with anATase activity having a higher substrate selectivity for 18:1-ACP thanfor 16:0-ACP according to the present invention can be that encoding anATase derived from organisms other than higher plants such as bacteria.When such a DNA sequence is used, appropriate expression regulatorysequences and a sequence encoding a transit peptide might be required inan appropriate arrangement upstream and/or downstream of the DNAsequence. Detailed constructions and procedures for generating sucharrangements can be found in laboratory manuals such as Molecularcloning 2nd ed. (Sambrook et al eds.), Cold Spring Harbor LaboratoryPress, New York, 1989, and are obvious to those skilled in the art.

A DNA sequence encoding a polypeptide with an ATase activity having ahigher substrate selectivity for 18:1-ACP than for 16:0-ACP for use inthe present invention can also be one encoding a derivative of ATasesdescribed above. In this context "derivative" means a polypeptide withone or more amino acid substitutions, deletions, insertions or additionsto any of the ATases described above, provided the change(s) in theamino acid sequence does not impair the ATase activity nor the substrateselectivity for 18:1.

A list of chilling-sensitive higher plants suitable for practice of thepresent invention to form transgenic plants includes, but is not limitedto, rice, maize, yam, sweet potato, cucumber, green pepper, eggplant,squash, banana, melon, kalanchoe, cyclamen, lily, rose, castor bean,sponge cucumber and tobacco.

Introduction of the DNA sequence into higher plants can be accomplishedby any of the established methods for plant transformation, such as theTi plasmid vector system of Agrobacteirum and electroporation ofprotoplasts (for example, see Plant genetic transformation and geneexpression; a laboratory manual (Draper, J. et al. eds.), BlackwellScientific Publications, 1988), in accordance with the target plant. Ingeneral, use of the Ti plasmid vector is preferred for dicotyledonousplants and physical methods such as electroporation are preferred formonocotyledonous plants and dicots that are not susceptible toAgrobacterium infection. Plant materials to be transformed can be anyexplants such as leaf disks, stem disks, tuber disks, protoplasts,callus, pollens or pollen tubes, in accordance with the transformationprotocol.

According to the present invention, introduction and expression in ahigher plant of a DNA sequence encoding an ATase that has a highersubstrate selectively for 18:1-ACP than for 16:0 increases theunsaturated fatty acid content particularly in PG and also results in aprominent decrease of saturated PG molecular species.

ATase catalyzes the first step of PG biosynthesis; at the same time,however, this step is common to the synthetic pathways of other lipidsin chloroplasts of many plants (see Background of the Invention) andthus the reaction products of ATase are utilized not only for PG butvarious other lipids. Furthermore, since the intrinsic ATase(s) is noteliminated in a transgenic plant expressing a foreign ATase with adifferent substrate selectivity, the foreign ATase has to compete withthe intrinsic ATase. For these reasons it was not possible to predictwhether the fatty acid composition of membrane lipids, much less themolecular species composition of PG, would change by expressing theforeign ATase. A prominent decrease of saturated PG molecular speciessuch as observed according to the present invention was totallyunexpected.

According to the present invention, it is possible to significantlydecrease the amount of unsaturated PG molecular species, the lipidspecies that gives rise to the phase separation of biomembranes andinduce the chilling injury of higher plants. This is the first case ofplant genetic engineering for chilling resistance.

The following examples illustrate and describe in more detail thepresent invention.

Experimental Example 1 Isolation of an Arabidopsis genomic DNA fragmentcoding for ATase

(1) Construction of a genomic DNA library

Genomic DNA was prepared from about 10 g (wet weight) of leaves andstems of Arabidopsis thaliana Heynhold (Lansberg strain) as described inCurrent Protocols in Molecular Biology (Ausbel, F. M. et al. eds.) vol.1, pp. 2,3,1-2,3,3, John Wiley and Sons, 1987.

The genomic DNA was partially digested with a restriction enzyme Sau3AI.inserted into the BamHI site of a lambda phage vector λDASH (Stratagene)and packaged in vitro using an in vitro packaging kit (GIGAPACK GOLD;Stratagene) to give a genomic DNA library in λ phage.

(2) Isolation of the ATase genomic DNA fragment

Escherichia coli strain P2392 (Stratagene) was infected with the phagelibrary, and three plates (10 cm×14 cm) with 6×10³ -6×10⁴ plaques eachwere screened. The phages were transferred to filters, which wereincubated at 68° C. for 2 hours in a hybridization solution containing5×Denhart's solution [0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1%bovine serum albumin], 6×SSC [900 mM NaCl, 90 mM sodium citrate, pH7.4], 10% dextran sulfate, 0.1% SDS and 100 μg/ml of salmon sperm DNA.

A 1.4 kb cDNA fragment for the ATase of squash (Cucurbita moschata Duch)was obtained by excising, with a restriction enzyme EcoRI, from therecombinant plasmid pAT-03 carrying the cDNA for the squash ATaseisolated from E. coil AT-03 (FERM BP-3094). This cDNA fragment wassubjected to nick translation (nick translation kit; Talcam Shuzo) with³² P-dATP to give a probe with a specific activity of about 10⁸ dpm/μg.

The probe was added to the hybridization solution and the filters werefurther incubated in this solution at 50° C. for 12 hours. Filters werethen washed at 40° C. with 2×SSC and 0.1% SDS solution and subjected toautoradiography to select phages which hybridized strongly to the probe.

The genomic DNA of Arabidopsis was excised from the phage DNA with arestriction enzyme BamHI and subjected to 0.8% agarose gelelectrophoresis to recover a 2.6 kb DNA fragment. This fragment stronglyhybridized to the probe. It was subcloned to a plasmid vectorpBLUESCRIPT (Stratagene) to give a plasmid pBB2.6.

Experimental Example 2 Isolation of the Arabidopsis ATase cDNA

(1) Construction of a cDNA library

Total RNA was prepared from about 15 g (wet weight) of leaves and stemsof Arabidopsis thaliana Heynhold (Lansberg strain) according to themethod described in Current Protocols in Molecular Biology (Ausbel, F.M. et al. eds.) vol. 1, pp. 4,3,1-4,3,4, John Wiley and Sons, 1987.Poly(A)⁺ RNA was prepared from the total RNA according to Wolf et al.(Nucleic Acids Res., 15:2911, 1987).

DNA complementary to the above poly(A)⁺ RNA was synthesized according tothe manual of a cDNA synthesis kit purchased from Pharmacia (Code No.27- 9260-01), using an oligo(dT) nucleotide as a primer. An EcoRIadapter containing a NotI recognition sequence (Pharmacia) was ligatedat each terminus of the double strand cDNA thus synthesized, which wasfollowed by ligation to the EcoRI site of a λ phage vector λZAPII(Stratagene). The phage DNA was packaged in vitro using an in vitropackaging kit (GIGAPACK H GOLD: Stratagene) to give a cDNA library inλZAPII.

(2) Isolation of the ATase cDNA

Escherichia coil strain XL1-Blue (Stratagene) was infected by the λphage library, and five plates (10 cm×14 cm) with 2×10⁴ plaques eachwere screened. The phages were transferred to filters, which wereincubated at 65° C. for 1 hour in a hybridization solution containing6×SSC, 0.05% skim milk and 0.02% sodium azide. A fragment of the genomicATase gene of Arabidopsis was obtained by excising, with a restrictionenzyme BamHI, from the recombinant plasmid pBB2.6 carrying a fragment ofthe Arabidopsis ATase gene (Experimental example 1(2)). This DNAfragment was subjected to nick translation (nick translation kit, TakaraShuzo) with ³² P-dATP to give a probe with a specific activity of about10⁷ dpm/μg.

The probe was added to the hybridization solution and the filters werefurther incubated in this solution at 65° C. for 16 hours. The filterswere then washed at 65° C. with 1×SSC and 0.1% SDS solution andsubjected to autoradiography to select phages which hybridized stronglyto the probe.

Inserts were excised from the phage DNAs with a restriction enzyme EcoRIand subjected to 1% agarose gel electrophoresis to determine the size ofthe fragments. One of the DNA fragments was about 1.4 kb. This fragmentwas subcloned in a plasmid vector pBLUESCRIPT (Stratagene) to give aplasmid pARAT. The nucleotide sequence of the fragment was determined bythe dideoxy termination method (Proc. Natl. Acad. Sci. USA 84:4767,1987).

The insert was 1,445 bp in length with an open reading frame of 1,380 bp(with a stop codon), which is shown in the sequence listing as SEQ IDNO: 1. In consideration of a high homology of the open reading frame tothe squash ATase cDNA in both the nucleotide and amino acid sequences,it was deduced that the DNA sequence from nucleotide 16 to 1392 of SEQID NO: 1encodes the precursor of the Arabidopsis ATase (SEQ ID No: 2)containing a transit peptide to chloroplasts, consisting of 459 aminoacids with a molecular mass of 50,431. Non-coding regions of 15 bp and53 bp were present at the upstream and downstream of the open readingframe, respectively. The amino acid sequence -90 to -1 in SEQ ID NO: 2ispresumably a transit peptide to chloroplasts by comparison with thesquash ATase.

Experimental Example 3 Expression of the ATase genes of Arabidopsis andsquash (control) in E. coil and the comparison of their substrateselectivities

(1) Construction of E. coil expression vectors

The plasmid pARAT obtained in Experimental Example 2(2) was digestedwith restriction enzymes HgaI, which cuts after nucleotide 285 of SEQ IDNO: 1, and EcoRI (the restriction site for which is in the vectorsequence downstream of the cDNA). The resulting 1.1 kb fragmentcontaining the Arabidopsis ATase cDNA was isolated from a low meltingagarose gel and made blunt-ended with the Klenow fragment. Meanwhile,plasmid pET3c (Novagen) was digested with a restriction enzyme BamHI andmade blunt-ended with the Klenow fragment, and then the phosphoryl groupat 5'-terminus was removed with bacterial alkaline phosphatase. The cDNAfragment of the Arabidopsis ATase and pEF3c thus obtained were ligatedby T4 DNA ligase to give an expression vector plasmid pAR1 containing aT7 promoter, a T7 leader sequence, the ATase cDNA of Arabidopsis, and aT7 terminator.

Plasmid pAT-03 containing a cDNA for the squash ATase was prepared fromE. coli AT-03 CFERM BP-3094), digested with restriction enzymes EcoRIand NaeI and then subjected to an electrophoresis on a low meltingagarose gel to isolate a 1.2 kb cDNA fragment of the squash ATase. Thisfragment was made blunt-ended with the Klenow fragment and thephosphoryl group at 5'-terminus was removed with bacterial alkalinephosphatase. The cDNA fragment of the squash ATase and pET3c thusobtained were ligated by T4 DNA ligase to give an expression vectorplasmid pSQ1 containing a T7 promoter, a T7 leader sequence, the ATasecDNA of squash and a T7 terminator.

Competent cells of Escherichia coil BL21 (DE3) (Novagen) were preparedas described in Molecular Cloning (Maniatis, T. et al. eds.), pp.250-251, 1982. Either of the plasmid pAR1 or pSQ1 obtained above wasintroduced into competent cells, and selection with ampicillin gavetransformants BLAR1 and BLSQ1, respectively.

The transformants BLAR1 and BLSQ1 were each inoculated into 500 ml ofthe LB medium (containing 200 μg/ml of ampicillin) and cultured at 37°C. Cells were grown until the turbidity of the culture reached 0.5OD. ata wavelength of 600 nm. Then isopropyl-thio-galactoside was added to afinal concentration of 0.4 mM, and the culture was continued for 3 hoursto induce the expression of the ATase protein. Bacterial cells werecollected from the culture by centrifugation at 14,000 g for 10 minutes.The pellets were rinsed with 50 mM Tris-HCl (pH 7.4) and resuspended inHM buffer [45 mM Tris-HCl, pH 7.4, 2 mM DTT, 10% glycerol, 10 mM sodiumascorbate, 1 mM benzamidine-HCl, 10 μg/ml leupeptin, 5 mM6-aminohexanoic acid]. The bacterial suspension was passed through aFrench pressure cell at 10,000 psi to break the cells. The homogenatewas centrifuged at 16,000 g for 10 minutes and further at 100,000 g for60 minutes, and the supernatant was recovered as a crude enzymefraction. The etude enzyme fraction was subjected to SDS electrophoresison a 10% polyacrylamide gel and stained with Coomassie-Brilliant Blue todetect the ATase of Arabidopsis or squash as a protein with a relativemolecular mass of about 40,000.

(2) Assay of the ATase activity

The ATase activity of the crude enzyme fractions prepared above wasassayed by the method of Nishida et al. (Plant Cell Physiol., 28:1071,1987) using 16:0-CoA and L-[U-¹⁴ C] glycerol 3-phosphate as thesubstrates. Both of the crude enzyme fractions from the E. colitransformants BLAR1 and BLSQ1 exhibited the ATase activity (the transferof 16:0 to glycerol 3-phosphate). The specific activities of ATase inthe fractions were 2,000 and 530 nmol/min. mg protein, respectively.

The substrate selectivity of the ATase activity thus obtained wasanalyzed according to Frentzen et al. (Plant Cell Physiol. 28:1195,1987). The reaction mixture contained 30 mM of glycerol 3-phosphate, 1.5μM each of [1-¹⁴ C] 16:0-ACP and [1-¹⁴ C] 18:1-ACP, and the crude enzymefraction of the expressed ATase corresponding to the enzyme activity ofabout 180 pmol/min. The selectivity was assayed at pH 7.4 and 8.2. Theresults are shown in Table 1. The expressed Arabidopsis ATase, incontrast to the expressed squash ATase, showed a high selectivity for18:1-ACP.

                  TABLE 1                                                         ______________________________________                                        Substrate selectivity of ATases expressed in E. coli                                    Incorporation into lyso-phosphatidic acid*                                    18:1/16:0                                                           Source of cDNA  pH 7.4  pH 8.2                                                ______________________________________                                        Arabidopsis     73/27   65/35                                                 Squash          68/32   56/44                                                 ______________________________________                                         *under the presence of 30 mM glycerol 3phophate, 1.5 μM [1.sup.14 C]       18:1ACP and 1.5 μM [1.sup.14 C] 16:0ACP.                              

EXAMPLE 1 Expression of the cDNA for Arabidopsis ATase in transgenictobacco

Tobacco is a chilling-sensitive plant, but relatively chilling-resistantamong sensitive plants. The cDNA for the Arabidopsis ATase wasintroduced and expressed in transgenic tobacco plants as follows.

(1) Construction of a plant expression vector

A plant binary expression plasmid pBI121 (Clontech) was digested withrestriction enzymes SacI and BamHI, made blunt-ended with the Klenowfragment, and ligated with T4 DNA ligase. Plasmid pBI121(-GUS) thusobtained has the β-glucuronidase (GUS) gene deleted. This plasmid hasunique cloning sites of XbaI and BamHI between the cauliflower mosaicvirus 35S promoter (hereinafter called 35S promoter) and the nopalinesynthase (NOS) terminator.

Plasmid pARAT, obtained in Experimental Example 2, was digested withEcoRI, and the 1.4 kb Arabidopsis ATase cDNA fragment and the vectorfragment were separated by a low-melting-point agarose gelelectrophoresis. The cDNA fragment was excised from the gel, purified,and was filed-in with the Klenow fragment. The cDNA fragment was clonedinto the filled-in Xbal site of pBI121(-GUS) obtained above to constructan expression plasmid pBI121-35SART, which carries the Arabidopsis ATasecDNA under the control of the 35S promoter and the NOS terminator.

(2) Introduction of pBI121-35SART into Agrobacterium

Agrobacterium tumefaciens LBA4404 (Clontech) was inoculated into 50 mlof YEB medium [beef extract 5 g/l, yeast extract 1 g/l, peptone 1 gA,sucrose 5 g/l 2 mM MgSO₄, pH 7.4] and harvested after a 24 hr culturingat 28° C. by centrifuging at 3,000 rpm, 4° C. for 20 minutes. The cellswere washed three times with 10 ml of 1 mM HEPES (pH 7.4), washed oncewith 3 ml of 10% glycerol, and suspended in 3 ml of 10% glycerol to beused in the following experiment.

50 μl of the Agrobacterium suspension and 1 μg of the plasmidpBI121-35SART were put into a cuvette and subjected to an electric pulseusing Gene Pulser electroporator (Bio-Rad) under the condition of 25 μF,2,500 V, 200 Ω to introduce the plasmid into the bacteria. Theelectropointed suspension was transferred to an Eppendorf tube and 800μl of SOC medium [triptone 20 g/l, yeast extract 5 g/l, NaCl 0.5 g/l,2.5 mM KCl, pH 7.0] was added, and the robe was kept at 28° C. for 1.5hours. 50 μl of the bacterial suspension was spread onto a YEB plate(agar 1.2%) containing 100 ppm of kanamycin and incubated at 28° C. for2 days.

A single colony was picked up from the colonies formed on the plate. Thecolony was cultured in a mall scale and the plasmid DNA was isolated bythe alkaline method. The plasmid DNA was digested with appropriaterestriction enzymes, separated on a 1% agarose gel, and the presence ofpBI121-355ART was confirmed by Southern blotting analyses using a ³²P-labelled Arabidopsis ATase cDNA fragment as the probe. ThisAgrobacterium was termed ALBSART.

(3) Transformation of tobacco

ALBSART was cultured in LB liquid medium containing 50 ppm of kanamycinfor 12 hours at 28° C. Cells were harvested from a 1.5 ml portion of theculture by centrifuging at 10,000 rpm for 3 minutes, washed with 1 ml ofLB medium to remove kanamycin, and was suspended in 1.5 ml of LB mediumto be used in the following experiment.

Young tobacco leaves were immersed in 0.5% NaClO for 10 minutes, washedthree times with sterile water, and excess water was wiped off withsterile filter paper. The leaves were aseptically cut into 1 cm² piecesand floated on the ALBSART suspension with the reverse side up for 2minutes with mild shaking, and excess bacterium suspension was wiped offon sterile filter paper. 1 ml of a tomato suspension culture (cultivar"Kurikoma") was spread on a MS-B5 plate [MS medium containingbenzyladenine 1.0 ppm, naphthalene acetate 0.1 ppm, agar 0.8%](Murashige, T. and Skoog, F. S., Plant Physiol., 15:473, 1962). A pieceof Whatman No. 1 filter paper (φ7 cm) was put on the tomato suspensionculture, and the tobacco leaf pieces were put on the filter paper withthe reverse side up. The plate was sealed with Parafilm® and incubatedat 25° C. for two days under 16 hour light/8 hour dark condition (exceptotherwise described, the tobacco explants/plants were incubated underthis condition). The leaves were transferred to a MS-B5 plate containing250 ppm Claforan (Hoechst) and further incubated for 10 days toeliminate Agrobacteria. The leaves were then put on MS-B5 mediumcontaining 250 ppm Claforan and 100 ppm kanaxnycin and incubated for 7days, during which period the rim of the leaf pieces formed callus andshoot primodiums. After another 10 days of incubation, the elongatedshoots were transferred to MS hormone free medium containing 250 ppmClaforan and 100 ppm kanamycin. Shoots that rooted on this medium within10 days of incubation were picked up as kanamycin-resistanttransformants and transferred to MS hormone free medium containing 250ppm Claforan in transparent plastic containers.

(4) Western blot analyses of the transformed tobacco plants

0.5 g (wet weight) of tobacco leaf samples were homogenized with mortarand pestle in an extraction buffer containing 80 mM Tris-HCl (pH 6.8),2% SDS, 5% glycerol, and 720 mM 2-mercaptoethanol. The homogenate wastransferred to an Eppendorf tube and heated at 100° C. for threeminutes, after which the supernatant was recovered by a centrifugationat 15,000 rpm, 20° C. for 10 minutes to obtain a crude total proteinextract. The protein concentration was measured using a protein assaykit (Bio-Rad) and adjusted to 1 μg/μl.

10 μl of the total protein extract was mixed with the sample loadingbuffer and electophoressed in an SDS-PAGE gel (Daiichi-kagaku Co.)according to Laemmli (Nature, 227:680, 1970). The proteins were blottedonto a PVDF membrane filter (Millipore) using an electroblottingapparatus (Atto) in a blotting buffer containing 0.025 M Tris, 0.192 Mglycine, 20% ethanol and 0.01% SDS at 100 V for one hour.

The membrane was immersed in the milk solution [5% skim milk (Difco), 10mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, 0.05% NAN₃, pH 7.2] and washedby shaking at room temperature for 3×10 minutes. It was furtherincubated in the milk solution at room temperature for three hours toblock the non-specific absorption of the antibody and then washed byshaking for 2×3 minutes in TBS-T buffer [10 mM Tris-HCl, 150 mM NaCl,0.05% Tween-20, 0.05% NaN₃, pH 7.2].

The membrane was incubated in an anti-(Arabidopsis ATase) mouseantiserum diluted 500-fold with TBS-T buffer with shaking at roomtemperature for two hours, followed by washing in the milk solution for3×10 minutes and in TBS-T buffer for 2×3 minutes at room temperature.Proteins reacted with the antibody were visualized by the peroxidasestaining using Vectastain ABC kit (Vector Laboratories) according to thesupplier's instruction.

The total proteins extracted from transformed tobacco plants contained aprotein reactive with the antibody against Arabidopsis ATase in anamount of approximately 0.5% of the total proteins.

EXAMPLE 2 Fatty acid composition of phosphatidylglycerol from the leavesof transgenic tobacco

Phosphatidylglycerol (PG) was extracted from the leaves of transgenictobacco plants obtained above and control non-transformed tobacco plantsto analyze the fatty acid composition.

(1) Extraction of total lipids

Total lipids were extracted according to Bligh and Dyer (Can. J.Biochem. Physiol., 37:911, 1959). 2 g (wet weight) of leaf samples werecut into strips using a scalpel and put quickly into 5 ml of pre-heated(80° C.) isopropanol containing 0.1% butylhydroxytoluene, kept at 80° C.for 5 minutes, and cooled to room temperature. 20 ml ofchloroform:methanol (1:2 v/v) was added and the leaves were homogenizedwith a homogenizer and let stand for 15 minutes. 12 ml each ofchloroform and distilled water were added and the mixture was vigorouslyshaken and then centrifuged at 3,000 rpm, 4° C. for 30 minutes toseparate it into aqueous and organic layers. The organic (bottom) layerwas recovered and, after adding an appropriate amount of ethanol,evaporated to dryness under reduced pressure at 30 ° C. using a rotaryevaporator. Total lipids thus obtained were dissolved in 2 ml ofchloroform:methanol (1:4, v/v).

(2) Fractionation of lipid classes

25 ml suspension of DEAE-Toyopearl (Toso) was mixed with 25 ml of 1 Msodium acetate (pH 7.0) to activate the resin. It was then washed withdistilled water, methanol, suspended in methanol and packed in a column(φ2 cm) to a height of 1.5 cm. The column was washed with 50 ml ofchloroform:methanol (1:4, v/v).

The total lipids were loaded onto the column. First,monogalactosyldiacytglycerol, digalactosyidiacylglycerol,phosphatidylethanolamine and phosphatidylcholine were eluted out with 50ml of chloroform:methanol (1:4, v/v). Second, phosphatidylserine waseluted out with 5 ml of acetic acid. Finally, PG,sulfoquinovosyldiacylglycerol and phosphatidylinositol were eluted with50 ml of chloroform:methanol:10 M aqueous ammonium acetate (20:80:0.2,v/v). After adding 15 ml of ethanol, the last fraction was evaporatedunder reduced pressure and the residue was dissolved in 1 ml ofchloroform:methanol (2:1, v/v).

PG was purified from the fractionated lipids by TLC on a silica gelplate (Merck #5721) using chloroform:acetone:methanol:acetic acid:water(50:20:10:15:5, v/v) as the developing solvent. The lipids werevisualized by primulin fluorescence and PG was identified by comparingthe migration rate with a standard PG preparation.

(3) Fatty acid analysis

Silica powder containing the PG was scraped off from the TLC plate andput into a screw-capped test robe. 2.5 ml of 5% HCl/methanol was addedto the tube and the lipid was methanolyzed at 85° C. for 2.5 hours inthe robe tightly capped. The resultant fatty acid methyl esters wereextracted four times with 5 ml of hexane, combined and concentratedunder reduced pressure, and analyzed by gas chromatography. Fatty acidswere identified by comparing the retention time with standard fatty acidmethyl esters and quantified with Shimadzu Chromatopack C-R2AX. Theresults are shown in Table 2.

While the content of saturated fatty acids (16:0+16:1t+stearic acid(18:0)) in PG was 68=1% in the control non-transformed plants, it wasdecreased to 63±1 % in the transgenic plants expressing the ArabidopsisATase. Considering that the sn-2 position of PG is occupied exclusivelyby 16:0 and 16:1t, the content of saturated molecular species in PG iscalculated from the fatty acid content to be 36±1% for non-transformedplants and 26±1% for transgenic plants (Table 2).

No significant difference was observed between the control and thetransgenic plants in the fatty acid compositions of major lipid classesother than PG.

                  TABLE 2                                                         ______________________________________                                        Fatty acid and molecular species compositions in PG                                           16:0 + 16:1t +                                                                            Saturated                                         Plant           18:0        molecular species                                 ______________________________________                                        Non-transformed tobacco                                                                       68 ± 1%  36 ± 1%                                        Arabidopsis     60 ± 1%  20 ± 2%                                        Transgenic tobacco                                                                            63 ± 1%  26 ± 1%                                        ______________________________________                                    

EXAMPLE 3 Transport of the expressed Arabidopsis ATase to chloroplasts

Intact chloroplasts were prepared from the transgenic tobacco of Example1 and control non-transformed tobacco plants and the chloroplastproteins were analyzed.

(1) Preparation of intact chloroplasts

10 g (wet weight) of leaf samples were chopped with scissors and quicklyput into 30 ml of ice-cold homogenizing buffer [50 mM sodiumpyrophosphate, 1 mM MgCl₂, 1 mM EDTA. 2Na, 2 mM sodium isoaseorbate,0.1% bovine serum albumin. 330 mM sorbitol, pH 7.8]. The leaves weremildly broken by a Polytron® and filtered through four layers ofMilacloth. The filtrate was centrifuged at 2,000 g, 4° C. for 2 minutesto recover the pellet, which was completely suspended in 3 ml ofsuspension buffer [50 mM HEPES-NaOH, 330 mM sorbitol, pH 7.6] using abrush. Cell debris were removed by a centrifugation at 100 g, 4° C. for2 minutes and the chloroplast fraction were recovered by centrifuging at2,000 g, 4° C. for 2 minutes. The pellet was completely re-suspended in1 ml of the suspension buffer using a brush.

A tube of Percoll® gradient (from bottom to top: 80% 2.6 ml, 40% 12 ml.15% 5.4 ml) was prepared at 4° C. and let stand for a while. Thechloroplast suspension was loaded onto the gradient and centrifuged at7,000 g, 4° C. for 15 minutes. Intact chloroplasts were separated at theinterface between 80% and 40% Percoll as a green band, which was washedwith five volumes of the suspension buffer and recovered by centrifugingat 2,000 g, 4° C. for 5 minutes.

(2) Analysis of the total chloroplast proteins

The intact chloroplasts were suspended in 400 μl of the extractionbuffer and the total chloroplast proteins were extracted in the same wayas described in Example 1(4). 10 μg of the total chloroplast proteinswere subjected to the Western blot analysis as described in Example1(4). The result is shown in FIG. 1.

A band corresponding to the Arabidopsis ATase, which reacted with theantibody against Arabidopsis ATase, was detected in the totalchloroplast protein preparations from transgenic tobacco plants. Thisindicates that the Arabidopsis ATase expressed in the transgenic tobaccoplants was transported to tobacco chloroplasts.

Furthermore, a soluble protein fraction was prepared from the intactchloroplasts of transgenic plants and analyzed by Western blotting. Aband corresponding to the Arabidopsis ATase, which reacted with theantibody against Arabidopsis ATase, was detected in the solublechloroplast protein preparation (not shown), which indicates that thetransported Arabidopsis ATase was localized in the chloroplast stroma.

EXAMPLE 4 Expression of the squash ATase in tobacco plants

In order to examine in more detail the effect of the saturated molecularspecies content in PG on the chilling sensitivity of higher plants, thecDNA coding for the squash ATase was introduced and expressed in tobaccoplants to obtain transgenic plants containing more saturated PGmolecular species than those inherent to tobacco. As is shown in Table 1of Experimental example 3, the squash ATase does not have a substrateselectivity for 18:1-ACP and transfers both 18:1-ACP and 16:0-ACP inalmost the same proportion to the sn-1 position of glycerol 3-phosphate.

The squash ATase cDNA has been cloned by the applicant and itsnucleotide sequence is known to public (Ishizaki (Nishizawa), O. et al,FEBS Lett., 238:424, 1988). It can therefore be obtained by any ofappropriate methods utilized in the field of genetic engineering, suchas chemical DNA synthesis and PCR, according to the sequenceinformation.

When the squash ATase is expressed in transformed tobacco plants, theATase has to be transported to chloroplasts since it functions there(see Detailed Description of the Invention). To assure this transport,the DNA sequence encoding the transit peptide portion of the ArabidopsisATase (amino acid -90 to -1 in SEQ ID NO: 2) was fused in frame to theDNA sequence encoding the mature protein of the squash ATase.

pARAT (Experimental example 2(2)) was digested with restriction enzymesHgaI, which curs after nucleotide 285 of SEQ ID NO: 1, and XhoI (therestriction site for which is in the vector sequence upstream of thecDNA). The 320 bp fragment containing the DNA sequence encoding theArabidopsis transit peptide was isolated and made blunt-ended with theKlenow fragment. Meanwhile the plasmid pAT-03 carrying a squash ATasecDNA (lshizaki, O. et al, FEBS Lett., 238:424, 1988) was digested with arestriction enzyme EcoRI and the 1.4 kb fragment containing the squashATase cDNA was isolated, which was inserted into the EcoRI site of pUC19(Takara Shuzo) to make the plasmid pUC19/AT03. This plasmid waslinearized with NaeI and the ends were made blunt with the Klenowfragment.

The two DNA fragments were ligated together with T4 DNA ligase and aplasmid having one transit peptide DNA fragment inserted in a correctorientation with the squash ATase cDNA was selected, which was termedpSQAR. pSQAR codes for, from 5' to 3', (most likely a part of) squashATase transit peptide and the Arabidopsis ATase transit peptide fused inframe to the mature squash ATase, between which are the multi-cloningsites derived from pARAT. The fusion protein would be processed to aprotein identical to the mature squash ATase except for the substitutionof Leu for Pro at the second position from the N-terminus uponexpression and transportation to chloroplasts in transgenic plants.

An EcoRI fragment encoding the fusion protein was excised from pSQAR,made blunt-ended with the Klenow fragment, and inserted into the SmaIsite of the plant transformation vector pBI121 (Clontech) to obtainpBI121-35SSQAR. This vector plasmid carries the DNA sequence encodingthe fusion protein under the control of the 35S promoter and the NOSterminator, with the structural gone for GUS inserted between the fusionprotein region and the terminator.

Tobacco was transformed with pBI121-25SSQAR as described in Example 1(2)and (3) to obtain transgenic tobacco plants expressing the squash ATase.PG samples were enacted from the leaves of the transgenic plants andtheir fatty acid compositions were analyzed as in Example 2. The contentof saturated fatty acids (16:0+16:1t+18:0) was 72±1% and the saturatedmolecular species content was calculated to be 44%. This value washigher than the value of 36% for the non-transformed tobacco (see Table2) indicating the fatty acid composition of PG from the transgenictobacco was shifted to a chilling-sensitive type. For comparison, PGfrom squash leaves contain 64% saturated molecular species.

EXAMPLE 5 Effects of a low temperature treatment on the photosyntheticactivities of transgenic tobacco leaves

Photosynthesis is one of the most dominant and important biochemicalprocesses in higher plants, and the loss of its activity leads to thedamage of physiological activity of the whole plant. The loss ofphotosynthetic activity by a low temperature treatment is therefore agood indication of the chilling sensitivity of the plant.

Accordingly, photosynthetic oxygen evolution of leaves was comparedbefore and after a low temperature treatment for the transgenic tobaccoplants of Examples 1 and 4, as well as those transformed with the vectorpBI121.

Oxygen evolution of leaves was measured with a Clark-type oxygenelectrode assembled for the gas-phase measurement. A 8.5-10 cm² leafdisk was cut from an intact leaf and placed on a wetted sponge mat inthe temperature-controlled chamber of the leaf disk electrode unit(Hanzatech, LD2). White light from a 100 W tungsten lamp (Hanzatech,LS2) passed through a heat-cut off filter (Hoya, HA-50) was used as theactinic light for photosynthetic oxygen evolution (1,000 μE/m² /sec).The gas phase of the chamber was replaced with air containing 5% CO₂every 7 minutes. Oxygen evolution from the leaf disk was measured at 27°C. continuously for about 90 minutes, then the temperature of thechamber was lowered to 1° C. The leaf disk was kept at this lowtemperature under the same illumination for 4 hours, after which thetemperature was raised again to 27° C. and oxygen evolution was measuredas above. FIG. 2 shows the results obtained with the transgenic tobaccoof Examples I and 4, and the control tobacco transformed with the vectorpBI121, which is identical to non-transformed tobacco with respect tochilling sensitivity.

In both measurements before and after the chilling treatment, the oxygenevolution activities gradually increased to reach a plateau after sometime. The activities at that time were taken as the activities beforeand after the treatment, respectively, and their ratio was calculated asan indicator of the chilling sensitivity as shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Damage of photosynthesis at 1° C. in transgenic tobacco plants         Tobacco plant  Activity after treatment at 1° C.                       transformed with                                                                             (relative to that before treatment)                            ______________________________________                                        pBI121                                                                        #1             0.86                                                           #2             0.70                                                           #3             0.73                                                           Arabidopsis ATase cDNA                                                        #1             0.91                                                           #2             0.96                                                           #3             0.93                                                           #4             0.90                                                           Squash ATase cDNA                                                             #1             0.53                                                           #2             0.41                                                           ______________________________________                                    

While the photosynthetic oxygen evolution activity of the control pBI121transformed plants decreased to 70-86% of the original level by lowtemperature treatment at 1° C. for 4 hours, that of the ArabidopsisATase transgenic plants little decreased and retained 90-96% of theoriginal level after the treatment. This shows that the photosyntheticoxygen evolution activity of the Arabidopsis ATase transgenic plants ismore resistant to low temperature than the control. It is thus concludedthat the Arabidopsis ATase transgenic plants are more chilling-resistantthan the control.

On the other hand, the photosynthetic oxygen evolution activity of thesquash ATase transgenic plants decreased to 41-53% of the original levelby low temperature treatment at 1° C. for 4 hours, which decrease issignificantly larger than the control indicating that the squash ATasetransgenic plants become more chilling-sensitive than the control.

EXAMPLE 6 Effects of a low temperature treatment on transgenic tobaccoplants

The effects of a low temperature treatment on the whole plant of thetransgenic tobacco plants of Example 1 and control non-transformed andpBI121-transformed tobacco plants were examined.

The tobacco plants were grown in vitro in transparent plasticcontainers. Upper parts of the plants thus grown were cut and eachtransferred onto MS hormone free medium containing 250 ppm of Claforanin the plastic containers and grown for two weeks at 25° C. under 16 hlight/8 h dark condition. Within that period the explants rooted anddeveloped into plantlets with three to four fully expanded leaves.

The tobacco plants in the container were put into a growth chamber(Koito Kogyo Co.: KPS-2000) set at a temperature of 1° C. and kept therefor 10 days under a fluorescent lamp illumination of 100 μE/m² /sec. Theplants were then transferred to 25° C. (16 h light/8 h dark) for twodays and chilling injuries on the plants were observed. FIGS. 3A-3D showone of the transgenic tobacco plants expressing the Arabidopsis ATaseand a control pBI121-transformed tobacco before and after the chillingtreatment.

pBI121-transformed tobacco plants, as well as non-transformed plants,developed white spots on their leaves after the chilling treatment,which results from the decay of chloroplasts (ehlorosis) by thetreatment (FIGS. 3A-3D, upper plate). On the other hand, the ArabidopsisATase transgenic plants suffered little damage by the treatment (FIG. 3,lower plates) indicating that they are more chilling-resistant than thecontrol plants.

The above Examples conclusively demonstrate application of the presentinvention to the engineering of chilling resistance into achilling-sensitive higher plant by introducing and expressing an ATaseof chilling-resistant plants in the chilling-sensitive plant and thusdecreasing the saturated molecular species in its PG. The fact that thetobacco plants engineered to contain a higher amount of saturated PGmolecular species were more sensitive to chilling injury further provesthe relationship between the chilling sensitivity and the PG molecularspecies composition, indicating that the process to give chillingresistance to higher plants according to the present invention can bewidely applicable to variety of crop plants. This is the first case ofplant genetic engineering for chilling resistance, which will invaluablycontribute to agricultural production in areas under chilling climate.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1445 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                            (B) LOCATION: 16..1392                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 16..285                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 286..1392                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ACCAAACACGCTTTAATGACTCTCACGTTTTCCTCC TCCGCCGCAACCGTT51                        MetThrLeuThrPheSerSerSerAlaAlaThrVal                                          -90-85-80                                                                     GCCGTTGCTGCTGCAACCGTAACCTCC TCCGCTAGGGTTCCGGTTTAT99                           AlaValAlaAlaAlaThrValThrSerSerAlaArgValProValTyr                              -75-70-65                                                                     CCACTCGCTTCGTCGACTCTTCGT GGATTAGTATCTTTCAGATTAACC147                          ProLeuAlaSerSerThrLeuArgGlyLeuValSerPheArgLeuThr                              -60-55-50                                                                     GCGAAGAAGCTGTTTCTGCCGCCTC TTCGTTCTCGCGGCGGCGTTAGT195                          AlaLysLysLeuPheLeuProProLeuArgSerArgGlyGlyValSer                              -45-40-35                                                                     GTGAGAGCCATGTCTGAGCTAGTTCAAGA TAAAGAATCGTCCGTCGCG243                          ValArgAlaMetSerGluLeuValGlnAspLysGluSerSerValAla                              -30-25-20-15                                                                  GCGAGCATTGCTTTCAATGA AGCCGCCGGTGAGACGCCGAGTGAGCTT291                          AlaSerIleAlaPheAsnGluAlaAlaGlyGluThrProSerGluLeu                              -10-51                                                                        AATCATTCCCGTACT TTCTTGGATGCGCGAAGTGAACAAGATCTTTTA339                          AsnHisSerArgThrPheLeuAspAlaArgSerGluGlnAspLeuLeu                              51015                                                                         TCTGGTATCAAGAAGGAA GCTGAAGCTGGAAGGTTGCCAGCAAATGTT387                          SerGlyIleLysLysGluAlaGluAlaGlyArgLeuProAlaAsnVal                              202530                                                                        GCAGCAGGAATGGAAGAATTGTAT TGGAACTACAAAAATGCAGTTTTA435                          AlaAlaGlyMetGluGluLeuTyrTrpAsnTyrLysAsnAlaValLeu                              35404550                                                                      AGTAGTGGAGCTTCCAGG GCAGATGAAACTGTTGTATCAAACATGTCT483                          SerSerGlyAlaSerArgAlaAspGluThrValValSerAsnMetSer                              556065                                                                        GTTGCTTTTGATCGC ATGCTTCTTGGTGTGGAGGATCCTTATACTTTT531                          ValAlaPheAspArgMetLeuLeuGlyValGluAspProTyrThrPhe                              707580                                                                        AATCCATATCATAAA GCAGTCAGAGAACCATTTGACTACTACATGTTT579                          AsnProTyrHisLysAlaValArgGluProPheAspTyrTyrMetPhe                              859095                                                                        GTCCATACATACATCCGT CCTCTTATTGATTTCAAAAATTCGTACGTT627                          ValHisThrTyrIleArgProLeuIleAspPheLysAsnSerTyrVal                              100105110                                                                     GGAAATGCTTCTATATTCTCTGAG CTGGAAGACAAGATTCGACAGGGA675                          GlyAsnAlaSerIlePheSerGluLeuGluAspLysIleArgGlnGly                              115120125130                                                                  CACAATATCGTGTTGATA TCAAACCATCAAAGTGAAGCTGATCCGGCT723                          HisAsnIleValLeuIleSerAsnHisGlnSerGluAlaAspProAla                              135140145                                                                     GTCATTTCTCTATTG CTTGAAGCACAATCTCCTTTCATAGGAGAGAAC771                          ValIleSerLeuLeuLeuGluAlaGlnSerProPheIleGlyGluAsn                              150155160                                                                     ATTAAATGTGTGGCT GGTGATCGAGTCATCACTGATCCTCTTTGTAAG819                          IleLysCysValAlaGlyAspArgValIleThrAspProLeuCysLys                              165170175                                                                     CCGTTCAGTATGGGAAGG AACCTCATATGTGTTTACTCGAAAAAGCAC867                          ProPheSerMetGlyArgAsnLeuIleCysValTyrSerLysLysHis                              180185190                                                                     ATGAATGTTGATCCTGAGCTTGTT GACATGAAAAGAAAAGCAAACACA915                          MetAsnValAspProGluLeuValAspMetLysArgLysAlaAsnThr                              195200205210                                                                  CGAAGCTTAAAGGAGATG GCTACAATGCTAAGGTCTGGCGGTCAACTT963                          ArgSerLeuLysGluMetAlaThrMetLeuArgSerGlyGlyGlnLeu                              215220225                                                                     ATATGGATTGCACCA AGCGGTGGAAGGGACCGCCCGAATCCTTCTACT1011                         IleTrpIleAlaProSerGlyGlyArgAspArgProAsnProSerThr                              230235240                                                                     GGGGAATGGTTTCCT GCACCCTTTGATGCTTCTTCGGTAGACAACATG1059                         GlyGluTrpPheProAlaProPheAspAlaSerSerValAspAsnMet                              245250255                                                                     AGAAGACTGGTTGAACAT TCTGGCGCTCCTGGACATATATATCCAATG1107                         ArgArgLeuValGluHisSerGlyAlaProGlyHisIleTyrProMet                              260265270                                                                     TCTTTGCTTTGCTATGACATCATG CCCCCTCCACCCCAGGTTGAGAAA1155                         SerLeuLeuCysTyrAspIleMetProProProProGlnValGluLys                              275280285290                                                                  GAAATCGGAGAGAAAAGA TTAGTTGGGTTTCACGGTACTGGACTATCA1203                         GluIleGlyGluLysArgLeuValGlyPheHisGlyThrGlyLeuSer                              295300305                                                                     ATTGCTCCTGAAATC AACTTCTCAGACGTCACAGCAGACTGCGAGAGC1251                         IleAlaProGluIleAsnPheSerAspValThrAlaAspCysGluSer                              310315320                                                                     CCTAATGAGGCGAAA GAAGCATACAGCCAAGCTTTGTACAAGTCGGTG1299                         ProAsnGluAlaLysGluAlaTyrSerGlnAlaLeuTyrLysSerVal                              325330335                                                                     AATGAACAATACGAGATC TTAAACTCTGCGATTAAACACAGAAGAGGA1347                         AsnGluGlnTyrGluIleLeuAsnSerAlaIleLysHisArgArgGly                              340345350                                                                     GTAGAAGCATCAACTTCAAGGGTC TCTTTGTCACAACCTTGGAAT1392                            ValGluAlaSerThrSerArgValSerLeuSerGlnProTrpAsn                                 355360365                                                                     TAGTCTCTCGTTTTAGGGATACACAAACACAATCAATGGAAAAT ACTCAAAAA1445                    (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 459 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetThrLeuThrPheSerSerSerAlaAlaThrValAlaVal AlaAla                             -90-85-80-75                                                                  AlaThrValThrSerSerAlaArgValProValTyrProLeuAlaSer                              -70-65 -60                                                                    SerThrLeuArgGlyLeuValSerPheArgLeuThrAlaLysLysLeu                              -55-50-45                                                                     PheLeuProProLeuArgSerArgGlyGlyValSerValArgAla Met                             -40-35-30                                                                     SerGluLeuValGlnAspLysGluSerSerValAlaAlaSerIleAla                              -25-20-15                                                                     PheAsnGluAla AlaGlyGluThrProSerGluLeuAsnHisSerArg                             -10-515                                                                       ThrPheLeuAspAlaArgSerGluGlnAspLeuLeuSerGlyIleLys                              10 1520                                                                       LysGluAlaGluAlaGlyArgLeuProAlaAsnValAlaAlaGlyMet                              253035                                                                        GluGluLeuTyrTrpAsnTyrLysAsnAl aValLeuSerSerGlyAla                             404550                                                                        SerArgAlaAspGluThrValValSerAsnMetSerValAlaPheAsp                              556065 70                                                                     ArgMetLeuLeuGlyValGluAspProTyrThrPheAsnProTyrHis                              758085                                                                        LysAlaValArgGluProPheAspTyrTyrMetPheValHisThr Tyr                             9095100                                                                       IleArgProLeuIleAspPheLysAsnSerTyrValGlyAsnAlaSer                              105110115                                                                     IlePheSerG luLeuGluAspLysIleArgGlnGlyHisAsnIleVal                             120125130                                                                     LeuIleSerAsnHisGlnSerGluAlaAspProAlaValIleSerLeu                              135140 145150                                                                 LeuLeuGluAlaGlnSerProPheIleGlyGluAsnIleLysCysVal                              155160165                                                                     AlaGlyAspArgValIleThrAspPr oLeuCysLysProPheSerMet                             170175180                                                                     GlyArgAsnLeuIleCysValTyrSerLysLysHisMetAsnValAsp                              185190 195                                                                    ProGluLeuValAspMetLysArgLysAlaAsnThrArgSerLeuLys                              200205210                                                                     GluMetAlaThrMetLeuArgSerGlyGlyGlnLeuIleTrpIleAla                              215 220225230                                                                 ProSerGlyGlyArgAspArgProAsnProSerThrGlyGluTrpPhe                              235240245                                                                     ProAlaP roPheAspAlaSerSerValAspAsnMetArgArgLeuVal                             250255260                                                                     GluHisSerGlyAlaProGlyHisIleTyrProMetSerLeuLeuCys                              265 270275                                                                    TyrAspIleMetProProProProGlnValGluLysGluIleGlyGlu                              280285290                                                                     LysArgLeuValGlyPheHisGlyThrGlyLeuSe rIleAlaProGlu                             295300305310                                                                  IleAsnPheSerAspValThrAlaAspCysGluSerProAsnGluAla                              315320 325                                                                    LysGluAlaTyrSerGlnAlaLeuTyrLysSerValAsnGluGlnTyr                              330335340                                                                     GluIleLeuAsnSerAlaIleLysHisArgArgGlyValGluAla Ser                             345350355                                                                     ThrSerArgValSerLeuSerGlnProTrpAsn                                             360365                                                                    

What is claimed is:
 1. A transgenic higher plant characterized by thepresence in at least one of its lipid classes of a higher proportion ofunsaturated fatty acids than inherently present in species of said plantand by the presence in its cells of an exogenous DNA sequence encoding apolypeptide with a glycerol 3-phosphate acyltransferase activity havinga higher substrate selectivity for oleoyl-(acyl-carrier-protein) thanfor palmitoyl-(acyl-carrier-protein).
 2. A transgenic higher plantaccording to claim 1, wherein the polypeptide is a glycerol 3-phosphateacyltransferase of a chilling-resistant plant.
 3. A transgenic higherplant according to claim 2, wherein the chilling-resistant plant isspinach, pea, or Arabidopsis.
 4. A process for increasing theunsaturated fatty acid content in lipids of a higher plant speciescomprising introducing into the cells thereof an exogenous DNA sequenceencoding a polypeptide with a glycerol 3-phosphate acyltransferaseactivity having a higher substrate selectivity foroleoyl-(acyl-carrier-protein) than for palmitoyl-(acyl-carrier-protein).5. A process according to claim 4, wherein the polypeptide is a glycerol3-phosphate acyltransferase of a chilling-resistant plant.
 6. A processaccording to claim 5, wherein the chilling-resistant plant is spinach,pea, or Arabidopsis.
 7. A transgenic higher plant characterized byhaving a lowered critical temperature for chilling injury than thatinherent in species of said plant and by containing in the biomembranesof its cells a decreased proportion of saturated phosphatidylglycerolmolecular species and by the presence in its cells of an exogenous DNAsequence encoding a polypeptide with a glycerol 3-phosphateacyltransferase activity having a higher substrate selectivity foroleoyl-(acyl-carrier-protein) than for palmitoyl-(acyl-carrier-protein).8. A transgenic higher plant according to claim 7, wherein thepolypeptide is a glycerol 3-phosphate acyltransferase of achilling-resistant plant.
 9. A transgenic higher plant according toclaim 8, wherein the chilling-resistant plant is spinach, pea, orArabidopsis.
 10. A process for lowering the critical temperature forchilling injury of a higher plant species comprising decreasing thecontent of saturated phosphatidylglycerol molecular species in thebiomembranes of its cells and introducing into its cells an exogenousDNA sequence encoding a polypeptide with a glycerol 3-phosphateacyltransferase activity having a higher substrate selectivity foroleoyl-(acyl-carrier-protein) than for palmitoyl-(acyl-carrier-protein).11. A process according to claim 10, wherein the polypeptide is aglycerol 3-phosphate acyltransferase of a chilling-resistant plant. 12.A process according to claim 11, wherein the chilling-resistant plant isspinach, pea, or Arabidopsis.